1. Field of the Invention
This invention generally relates to the field of cross-linked hydrogel polymers formed into microgels for use in delivery of bioactive materials such as antigens, DNA and other therapeutics.
2. Description of the Related Art
There is a great need for the development of vaccines against AIDS, Hepatitis C, cancer and other diseases. Traditional vaccination strategies, based on live attenuated viruses, have been ineffective at generating vaccines against these diseases, largely as result of their high toxicity. Vaccines based on protein antigens are a new vaccination strategy that have considerable promise because of their low toxicity and widespread applicability. However, the clinical success of protein-based vaccines has been limited, due to delivery problems, and new protein delivery vehicles are needed that can enhance the efficacy of protein-based vaccines.
A key limitation of current protein-based vaccines is their inability to activate cytotoxic T lymphocytes (CTL). The activation of CTL is critical for the development of immunity against viruses and tumors. CTL are activated by antigen presenting cells (APCs) through the Class I antigen presentation pathway. APCs generally only present cytoplasmic proteins as Class I antigens, although Class I antigen presentation of proteins residing in phagosomes also occurs under certain circumstances (Jondal, M., Schirmbeck, R. & Reimann, J. (1996) Immunity 5, 295-302.
Microparticles, 0.2-5 μm in diameter, have recently gained interest as delivery vehicles for protein-based vaccines because of their ability to enhance the Class I antigen presentation of protein antigens (Oh, Yu-Kyoung, Harding, C. V.; Swanson, J. A.; Vaccine. 1997, (15), 511-518; Andrianov, et al., U.S. Pat. No. 5,529,777; and Staas, et al., U.S. Pat. No. 6,321,731). Two mechanisms have been proposed to explain the ability of microparticles to enhance the Class I antigen presentation of protein antigens. The first involves disruption of phagosomes by microparticles leading to release of protein antigens into the cytoplasm of APCs, where they are processed for antigen presentation as endogenous proteins. The second uses microparticles to deliver protein antigens to phagolysosomal compartments that contain MHC I receptors that are being recycled from the plasma membrane. Once delivered these proteins are subsequently degraded by phagolysosomal enzymes into antigenic peptides that complex MHC I receptors and are then trafficked to the cell surface for antigen presentation.
Protein therapeutics have tremendous clinical potential and are currently being investigated for the treatment of cancer, vaccine development and for manipulating the host response to implanted biomaterials. However, the effective utilization of protein therapeutics requires the development of materials that can deliver bioactive material to diseased tissues and cells. At present, the majority of protein delivery vehicles are based on hydrophobic polymers, such as poly(lactide-co-glycolide) (PLGA). See O'Hagan, D. et al., in U.S. Pat. Nos. 6,306,405 and 6,086,901, and in Adv. Drug Delivery Rev, 32, 225 (1998). However, PLGA based delivery vehicles have been problematic because of their poor water solubility. Proteins are encapsulated into PLGA based materials through an emulsion procedure that exposes them to organic solvents, high shear stress and/or ultrasonic cavitation. This procedure frequently causes protein denaturation and inactivation as shown by Xing D et al., Vaccine, 14, 205-213 (1996). Hydrogels and microgels have therefore been proposed as an alternative protein delivery vehicle because they can encapsulate the protein in a totally aqueous environment, under mild conditions. See Park, K. et al., Biodegradable Hydrogels for Drug Delivery; Technomic Publishing Co, Lancaster, Pa. (1993); Peppas. N. A. Hydrogels in Medicine and Pharmacy; CRC press: Vol II, Boca Raton, Fla., 1986; and Lee, K. Y. et al., Chemical Reviews, 101, 1869-179 (2001).
A key problem in the field of hydrogel research is the development of materials that can release their contents in response to pathological stimuli, allowing for the targeting of protein therapeutics to diseased tissues and cells. A particularly important pathological stimulus is mildly acidic pH. For example, tumors exist at acidic pHs between 6.4-6.8, and the phagolysosomes of phagocytic cells are at pHs between 4.5-5.0. The acidic nature of these compartments has stimulated a need for the development of hydrogels and microgels that can selectively release their contents under mildly acidic conditions.
A particularly important application of protein delivery systems is the development of particulate materials that can deliver proteins to phagocytic cells, such as antigen presenting cells. Micron sized protein loaded hydrogels (microgels) have been investigated for this purpose because they are small enough to be phagocytosed. At present, micron sized hydrogels have been synthesized using crosslinkers that do not degrade under biological conditions, and hence have had limited success in drug delivery applications.
Currently, hydrogels are synthesized using crosslinkers that contain either, amide, ester, or carbonate linkages. Sawhney, A. et al., Macromolecules, 26, 581-587 (1993), describe bioerodible hydrogels based on photopolymerized poly(ethyleneglycol)-co-poly(α-hydroxy acid) diacrylate macromers which utilize an ester linkage. Sheppard, R. C. et al., in U.S. Pat. No. 5,191,015, describe an insoluble polymer with contiguous cleavable crosslinkers and functional groups, wherein the crosslinking agent is an acid degradable ketal crosslinker. Sanxia. L, et al., describe release behavior of high molecular weight solutes from poly(ethylene glycol)-based degradable networks in Macromolecules, 33, 2509-2515 (2000). See also Dijk-Wolthius, W. N. E. et al., Macromolecules. 30, 4639-4645 (1997). A crosslinked network of poly-methylmethacrylate has been synthesized using an ethylene glycol di(1-methacryloyoxy)ethyl ether crosslinker that is breakable and pH-responsive, described by Ruckenstein E. et al., Macromolecules. 32, 3979-3983 (1999). But networks synthesized with this crosslinker only degrade under strong acidic conditions, such as pH 2.0 and below. The hydrolysis of this type of linkage is not acid catalyzed at mildly acidic levels present in biological applications. Thus, there is a need for hydrogels and microgels that degrade under mildly acidic conditions.